Fig. 4: Mkx⁺ cell ablation leads to compromised defect repair.

A Schematic for calvarial bone defect. Mkx^tdT or Mkx^tdT/iDTR mice were administered TM followed by local diphtheria toxin (DTX) overlying the skull, and then subjected to full-thickness frontal bone defects. Samples were harvested at day 28 post-defect. B μCT reconstructions of the defect site in a top-down view (above) and sagittal cross-sectional images (below). Margins of original defect are indicated by dashed red lines. C–G μCT quantification of bone healing among Mkx^tdT and Mkx^tdT/iDTR mice, including C bone volume (BV), D bone volume/tissue volume (BV/TV), E residual defect diameter, and F bone fractional area (BFA). G Hematoxylin and eosin (H&E) staining of coronal cross sections of the healing defect site from Mkx^tdT or Mkx^tdT/iDTR mice, d 28 after defect. Black arrowheads indicate original defect sites. Scale bar: 100 μm. H–J Immunostaining of OCN at the defect edge (H) and quantification of OCN (I) and percentage of Mkx^tdT cells among OCN⁺ cells within the defect site (J). GFP reporter activity not shown. K Immunostaining of CD31⁺ blood vessels at the defect edge from Mkx^tdT and Mkx^tdT/iDTR mice (left) and quantification (right). L Immunostaining of TUBB3⁺ (Beta III tubulin) nerve fibers at the defect edge from Mkx^tdT and Mkx^tdT/iDTR mice (left) and quantification (right). White dashed lines indicate healing bone edges. Scale bar: 100 μm. Dots in scatterplots represent values from individual measurement, whereas mean and 1 SD are indicated by crosshairs and whiskers. Relative staining: individual value was normalized to mean fluorescence intensity of control group (Mkx^tdT). *p < 0.05; ** p < 0.01. A two-tailed Student t-test was used for all comparisons.